This project will evaluate the properties of a novel glutathione peroxidase enzyme that is considerably enriched in lungs. This recently described protein (called 1-cys Peroxiredoxin or Peroxiredoxin 6, Prdx 6) represents the only non-selenium glutathione peroxidase of relatively high specific activity. An important characteristic in the spectrum activity for this enzyme is its ability, unlike classical glutathione peroxidase, to reduce phospholipid hydroperoxides. During the present grant Support as an R-01, we have demonstrated induction of Prdx 6 during hyperoxic stress and provided evidence that the enzyme protects against oxidant stress in cell culture and whole animal studies. Further, we have developed methods for isolation of Prdx 6 protein from rat and bovine lungs and have established expression systems for generation of active recombinant protein. We postulate that reduction of oxidized membrane phospholipids represents the basis for the anti-oxidant effect of the enzyme. Specific Aim I will utilize mouse models of altered Prdx 6 expression to continue our studies of the role of Prdx 6 in anti-oxidant defense. One of the models, Prdx 6 knock-out mice, was developed during the present period of grant support. The second Specific Aim will investigate control of Prdx 6 expression with special emphasis on the anti-oxidant response element and Nrf 2. Specific Aim 3 will evaluate substrate-protein interactions with a special focus on phospholipid binding by the protein as a requirement for the phospholipid hydroperoxide peroxidase activity. These studies will utilize site directed mutagenesis based on deduction from the crystal structure of the enzyme. Specific Aim 4 will utilize cellular systems for analysis of the role of nGST in activation of Prdx 6 activity. The proposed studies will provide a coordinated effort to investigate the role of this novel anti-oxidant enzyme in lung defense against oxidant stress and will provide new information concerning the biochemical regulation of its enzymatic activity.